The contractile properties of smooth muscle are broadly classified as phasic (fast) and tonic (slow). Phasic smooth muscle is characterized by a rapid rates of force activation, force relaxation and Vmax, whereas tonic smooth muscle is characterized by slow rates of force activation, force relaxation and Vmax. However, the molecular mechanism that regulates the contractile properties of smooth muscle is unknown. The overall goal of this grant is to determine the molecular mechanism for the contractile properties of smooth muscle and to elucidate the mechanism for fast and slow contractile properties. Our hypothesis is that splice variant isoforms of contractile proteins determine the contractile properties of smooth muscle. The specific aims to test this hypothesis are to determine if splice variants of MLC17 (Specific Aim 1), MHC (Specific Aim 2) and MLC phosphatase (Specific Aim 3) are molecular determinants of the contractile properties of smooth muscle. We will also determine if splice variants isoforms of MLC phosphatase effect either the magnitude or sensitivity of agonist induced force enhancement (Specific Aim 3). To test these Specific Aims we will force the expression of both splice variant isoforms of MLC17, MHC and MLC phosphatase in cultured embryonic aortic and gizzard smooth muscle cells. We will determine the effects of the expression of the isoform normally present or not expressed in the cultured aortic and gizzard smooth muscle cells on the mechanical properties of cultured smooth muscle cells. After forcing the expression of a single contractile protein, we will determine the maximum force, the rates of force activation and force relaxation, Vmax, and MLC20 phosphorylation of single cultured smooth muscle cells and compare the results to those obtained in the non-transfected controls. These experiments will elucidate the effects of the expression of a single contractile protein, in isolation, on the mechanical properties of cultured smooth muscle cells. The results of these studies should elucidate the mechanism that determines the contractile properties of smooth muscle, and form a foundation for future investigation of how smooth muscle contractility is altered by disease states.